This invention relates to novel mutant enzymes or enzyme variants useful in formulating detergent compositions in exhibiting improved wash performance, cleaning and detergent compositions containing said enzymes, mutated genes coding for the expression of said enzymes when inserted in a suitable host cell or organism and methods of selecting the amino acid residues to be changed in a parent enzyme in order to perform better in a given wash liquor under specified conditions.
In the detergent industry, enzymes have been implemented in washing formulations for more than 20 years. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. Commercially, proteases are most important.
Although proteases have been used in the detergent industry for more than 20 years, it is still not exactly known which physical or chemical characteristics are responsible for a good washing performance or ability of a protease.
The currently used proteases have been found by isolating proteases from nature and testing them in detergent formulations.
Bacillus Proteases
Enzymes cleaving the amide linkages in protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W.H. Freeman and Company, San Francisco, Chapter 3). Bacteria of the Bacillus species secrete two extracellular species of protease, a neutral, or metalloprotease, and an alkaline protease which is functionally a serine endopeptidase, referred to as subtilisin. Secretion of these proteases has been linked to the bacterial growth cycle, with greatest expression of protease during the stationary phase, when sporulation also occurs. Joliffe et al. (1980, J. Bacterial 141:1199-1208) has suggested that Bacillus proteases function in cell wall turnover.
Subtilisin
A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds and in which there is an essential serine residue at the active site (White, Handler and Smith, 1973 xe2x80x9cPrinciples of Biochemistry,xe2x80x9d Fifth Edition, McGraw-Hill Book Company, N.Y., pp. 271-272).
The bacterial serine proteases have molecular weights in the range of 20,000 to 45,000. They are inhibited by diisopropylfluorophosphate, but in contrast to metalloproteases, are resistant to ethylene diamino tetraacetic acid (EDTA) (although they are stabilized at high temperatures by calcium ions). They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more narrow term, alkaline protease, covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest, 1977, Bacteriological Rev. 41:711-753).
In relation to the present invention a subtilisin is a serine protease produced by Gram-positive bacteria or fungi. A wide variety of subtilisins have been identified and the amino acid sequence of a number of subtilisins have been determined. These include at least six subtilisins from Bacillus strains, namely, subtilisin 168, subtilisin BPNxe2x80x2, subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus and mesentericopeptidase (Kurihara et al., 1972, J. Biol. Chem. 247:5629-5631; Wells et al., 1983, Nucleic Acids Res. 11:7911-7925; Stahl and Ferrari, 1984, J. Bacteriol. 159:811-819, Jacobs et al., 1985, Nucl. Acids Res. 13:8913-8926; Nedkov et al., 1985, Biol. Chem. Hoppe-Seyler 366:421-430, Svendsen et al., 1986, FEBS Lett 196:228-232), one subtilisin from an actinomycetales, thermitase from Thermoactinomyces vulgaris (Meloun et al., 1985, FEBS Lett. 1983: 195-200) and one fungal subtilisin, proteinase K from Tritirachium album (Jany and Mayer, 1985, Biol. Chem. Hoppe-Seyler 366:584-492).
Subtilisins are well-characterized physically and chemically. In addition to knowledge of the primary structure (amino acid sequence) of these enzymes, over 50 high resolution X-ray structures of subtilisin have been determined which delineate the binding of substrate, transition state, products, at least three different protease inhibitors and define the-structural consequences for natural variation (Kraut, 1977, Ann. Rev. Biochem. 46:331-358).
In the context of this invention, a subtilisin variant or mutated subtilisin protease means a subtilisin that has been produced by an organism which is expressing a mutant gene derived from a parent microorganism which possessed an original or parent gene and which produced a corresponding parent enzyme, the parent gene having been mutated in order to produce the mutant gene from which said mutated subtilisin protease is produced when expressed in a suitable host.
Random and site-directed mutations of the subtilisin gene have both arisen from knowledge of the physical and chemical properties of the enzyme and contributed information relating to subtilisin""s catalytic activity, substrate specificity, tertiary structure, etc. (Wells et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84; 1219-1223; Wells et al., 1986, Phil. Trans. R. Soc. Lond. A. 317:415-423: Hwang and Warshel, 1987, Biochem. 26:2669-2673; Rao et al., 1987, Nature 328:551-554).
Especially site-directed mutagenesis of the subtilisin genes has attracted much attention, and various mutations are described in the following patent applications and patents:
EP Publ. No. 130756 (GENENTECH) (corresponding to U.S. Pat. No. 4,760,025 (GENENCOR)) relating to site specific or randomly generated mutations in xe2x80x9ccarbonyl hydrolasesxe2x80x9d and subsequent screening of the mutated enzymes for various properties, such as kcat/km ratio, pH-activity profile and oxidation stability. Apart from revealing that site-specific mutation is feasible and that mutation of subtilisin BPNxe2x80x2 in certain specified positions, i.e. xe2x88x921Tyr, 32Asp, 155Asn, 104Tyr, 222Met, 166Gly, 64His, 169Gly, 189Phe, 33Ser, 221Ser, 217Tyr, 156Glu or 152Ala, provide for enzymes exhibiting altered properties, this application does not contribute to solving the problem of deciding where to introduce mutations in order to obtain enzymes with desired properties.
EP Publ. No. 214435 (HENKEL) relating to cloning and expression of subtilisin Carlsberg and two mutants thereof. In this application no reason for mutation of 158Asp to 158Ser and 161Ser to 161Asp is provided.
In International Patent Publication No. WO 87/04461 (AMGEN) it is proposed to reduce the number of Asn-Gly sequences present in the parent enzyme in order to obtain mutated enzymes exhibiting improved pH and heat stabilities. In the application, emphasis is put on removing, mutating, or modifying the 109Asn and the 218Asn residues in subtilisin BPNxe2x80x2.
International patent publication No. WO 87/05050 (GENEX) discloses random mutation and subsequent screening of a large number of mutants of subtilisin BPNxe2x80x2 for improved properties. In the application, mutations are described in positions 218Asn, 131Gly, 254Thr, 166Gly, 116Ala, 188Ser, 126Leu and 53Ser.
In EP Application No. 87303761 (GENENTECH) it is described how homology considerations at both primary and tertiary structural levels may be applied to identify equivalent amino acid residues whether conserved or not. This information together with the inventors"" knowledge of the tertiary structure of subtilisin BPNxe2x80x2 led the inventors to select a number of positions susceptible to mutation with an expectation of obtaining mutants with altered properties. The positions so identified are: 124Met, 222Met, 104Tyr, 152Ala, 156Glu, 166Gly, 169Gly, 189Phe, 217Tyr. Also 155Asn, 21Tyr, 22Thr, 24Ser, 32Asp, 33Ser, 36Asp, 46Gly, 48Ala, 49Ser, 50Met, 77Asn, 87Ser, 94Lys, 95Val, 96Leu, 107Ile, 110Gly, 170Lys, 171Tyr, 172Pro, 197Asp, 199Met, 204Ser, 213Lys and 221Ser. The positions are identified as being expected to influence various properties of the enzyme. In addition, a number of mutations are exemplified to support these suggestions. In addition to single mutations in these positions, the inventors also performed a number of multiple mutations. Furthermore, the inventors identified 215Gly, 67His, 126Leu, 135Leu and amino acid residues within the segments 97-103, 126-129, 213-215 and 152-172 as having interest, but mutations in any of these positions are not exemplified.
EP Publ. No. 260105 (GENENCOR) describes modification of certain properties in enzymes containing a catalytic triad by selecting an amino acid residue within about 15xc3x85 from the catalytic triad and replacing the selected amino acid residue with another residue. Enzymes of the subtilisin type described in the present specification are specifically mentioned as belonging to the class of enzymes containing a catalytic triad. In subtilisins, positions 222 and 217 are indicated as preferred positions for replacement.
International Patent Publication No. WO 88/06624 (GIST-BROCADES NV) discloses the DNA and amino acid sequences of a subtilisin protease designated PB92 which is almost 100% homologous to the amino acid sequence of Subtilisin 309.
International Patent Publication No. WO 88/07578 (GENENTECH) claims mutated enzymes derived from a precursor enzyme by replacement or modification of at least one catalytic group of an amino acid residue. The inventors state that by doing so a mutated enzyme is obtained that is reactive with substrates containing the modified or replaced catalytic group (substrate-assisted catalysis).
The general theory is based on B. amyloliquefaciens subtilisin (BPNxe2x80x2), where modifications have been described in positions 64His that was modified into 64Ala alone or in combination with a xe2x80x9chelperxe2x80x9d mutation of Ser-24-Cys. Modifications are also suggested in the amino acid residues 32Asp and 221Ser and a xe2x80x9chelperxe2x80x9d mutation of Ala-48-Glu.
International Patent Publication No. WO 88/08028 (GENEX) discloses genetic engineering around metal ion binding sites for the stabilization of proteins. This publication also uses Subtilisin BPNxe2x80x2 as an example and points at the following amino acid residues as candidates for substitution 172Pro (P172D, P172E), 131Gly (G131D), 76Asn (N76D; N76D+P172D(E)), 78Ser (S78D). Further, suggestions are made for the combined mutants N76D+S78D+G131D+P172D(E); N76D+G131D; S78D+G131D; S78D+P172D(E) and S78D+G131D+P172D(E).
International Patent Publication No. WO 88/08033 (AMGEN) discloses a number of subtilisin analogues having a modified calcium binding site and either Asn or Gly replaced in any Asn-Gly sequence present in the molecule thereby obtaining enzymes exhibiting improved thermal and pH stability. One of the calcium binding sites is disclosed as involving the amino acid residues 41Asp, 75Leu, 76Asn, 77Asn, 78Ser, 79Ile, 80UGly, 81Val, 208Thr and 214Tyr; other potential calcium binding sites are suggested at 140Asp and 172Pro; 14Pro and 271Gln; and 172Pro and 195Glu or 197Asp. Also mutating the 109Asn and 218Asn positions is suggested. Mutants produced are N109S, N109S+N218S, N76D+N109S+N218S, N76D+N77D+N109S+N218S, N76D+I79E+N109S+N218S.
International Patent Publication No. WO 88/08164 (GENEX) describes a method for identifying residues in a protein which may be substituted by a cysteine to permit formation of potentially stabilizing disulfide bonds. The method is based on detailed knowledge of the three dimensional structure of the protein and uses a computer for selecting the positions. In relation to subtilisin proteases, Subtilisin BPNxe2x80x2 was used as a model system. Using the method on Subtilisin BPNxe2x80x2 resulted in the suggestion of 11 candidates for introducing disulfide bonds, i.e. 1:T22C+S87C, 2:V26C+L235C, 3:G47C+P57C, 4:M50C+N109C, 5:E156C+T164C, 6:V165C+K170C, 7:V165C+S191C, 8:Q206C+A216C, 9:A230C+V270C, 10:I234C+A274C and 11:H238C+W241C. Of these, four were produced, i.e. 1, 2, 4 and 8, of which two did not provide any stabilizing effect, i.e. 2 and 4. Further mutants were produced by combining two of these mutants with each other and one with another mutation, viz. T22C+S87C+N218S and T22C+S87C+Q206C+A216C. Also, a number of further unsuccessful mutants were produced, viz. AlC+S78C, S24C+S87C, K27C+S89C, A85C+A232C, I122C+V147C, S249C+A273C and T253C+A273C.
In addition, it has been shown by Thomas, Russell and Fersht, Nature 318, 375-376 (1985) that the exchange of 99Asp into 99Ser in subtilisin BPNxe2x80x2 changes the pH dependency of the enzyme.
In a subsequent article J. Mol. Biol. 193, 803-813 (1987), the same authors discussed the substitution of 156Ser for 156Glu.
Both of these mutations are within a distance of about 15 xc3x85 from the active 64His.
In Nature 328, 496-500 (1987) Russel and Fersht discuss the results of their experiments and present rules for changing pH-activity profiles by mutating an enzyme to obtain changes in surface charge.
Isoelectric Point (pIo)
The isoelectric point, pIo, is defined as the pH value where the enzyme molecule complex (with optionally attached metal or other ions) is neutral, i.e. the sum of electrostatic charges. (net electrostatic charge=NEC) on the complex is equal to zero. In this sum of course consideration of the positive or negative nature of the individual electrostatic charges must be taken into account.
The isoelectric point is conveniently calculated by using equilibrium considerations using pK values for the various charged residues in the enzyme in question and then finding by iteration the pH value where the NEC of the enzyme molecule is equal to zero.
One problem with this calculation is that the pK values for the charged residues are dependent on their environment and consequently subject to variation. However, very good results are obtainable by allocating specific approximate pK values to the charged residues independently of the actual value. It is also possible to perform more sophisticated calculations, partly taking the environment into consideration.
The pIo may also be determined experimentally by isoelectric focusing or by titrating a solution containing the enzyme. In addition, the various pK values for the charged residues may be determined experimentally by titration.
Industrial Applications of Subtilisins
Proteases such as subtilisins have found much utility in industry, particularly in detergent formulations, as they are useful for removing proteinaceous stains.
At present, the following proteases are known, many of which are marketed in large quantities in many countries of the world:
Subtilisin BPNxe2x80x2 or Novo, available from e.g. SIGMA, St. Louis, U.S.A.;
Subtilisin Carlsberg, marketed by Novo-Nordisk A/S (Denmark) as ALCALASE(copyright) and by IBIS (Holland) as MAXATASE(copyright);
A Bacillus lentus subtilisin, marketed by NOVO INDUSTRI A/S (Denmark) as SAVINASE(copyright);
Enzymes closely resembling SAVINASE(copyright) such as MAXACAL(copyright) marketed by IBIS and OPTICLEAN(copyright) marketed by MILES KALI CHEMIE (FRG);
A Bacillus lentus subtilisin, marketed by Novo Nordisk A/S (Denmark) as ESPERASE(copyright); and
KAZUSASE(copyright) marketed by SHOWA DENKO (Japan).
However, in order to be effective, these enzymes must not only exhibit activity under washing conditions, but also be compatible with other detergent components during detergent production and storage.
For example, subtilisins may be used in combination with other enzymes active against other substrates and, therefore, the selected subtilisin should possess stability towards and preferably should not catalyze the degradation of the other enzymes. In addition, the chosen subtilisin should be resistant to the action from other components in the detergent formulation, such as bleaching agents, oxidizing agents, etc., in particular an enzyme to be used in a detergent formulation should be stable with respect to the oxidizing power, calcium binding properties and pH conditions rendered by the non-enzymatic components in the detergent during storage and in the wash liquor during wash.
The ability of an enzyme to catalyze the degradation of various naturally occurring substrates present on the objects to be cleaned during e.g. wash is often referred to as its washing ability, washability, detergency or wash performance. Throughout this application the term wash performance will be used to encompass this property.
Naturally occurring subtilisins have been found to possess properties which are highly variable in relation to their washing power or ability under variations in parameters such as pH. Several of the above marketed detergent proteases, indeed, have a better performance than those marketed about 20 years ago, but for optimal performance each enzyme has its own specific conditions regarding formulation and wash conditions, e.g. pH, temperature, ionic strength (xe2x95x90I), active system (tensides, surfactants, bleaching agent, etc.), builders, etc.
As a result, it has been found that an enzyme possessing desirable properties at low pH and low I may be less attractive at more alkaline conditions and high I, or an enzyme exhibiting fine properties at high pH and high I may be less attractive at low pH, low I conditions.
The advent and development of recombinant DNA techniques has had a profound influence in the field of protein chemistry.
It has been envisaged that these techniques will make it possible to design peptides and proteins, such as enzymes and hormones according to desired specifications, enabling the production of compounds exhibiting desired properties.
It is now possible to construct enzymes having desired amino acid sequences, and as indicated above a fair amount of research has been devoted to designing subtilisins with altered properties. The proposals include the technique of producing and screening a large number of mutated enzymes as described in EP Publ. No. 130756 (GENENTECH) (U.S. Pat. No. 4,760,025 (GENENCOR)) and International patent Publ. No. WO 87/05050 (GENEX). These methods correspond to the classical method of isolating native enzymes and screening them for their properties, but is more efficient through the knowledge of the presence of a large number of different mutant enzymes.
Since a subtilisin enzyme typically comprises 275 amino acid residues each capable of being 1 out of 20 possible naturally occurring amino acids, one very serious drawback in that procedure is the very large number of mutations generated that has to be submitted to a preliminary screening prior to further testing of selected mutants showing interesting characteristics at the first screening, since no guidance is indicated in determining which amino acid residues to change in order to obtain a desired enzyme with improved properties for the use in question, such as, in this case formulating detergent compositions exhibiting improved wash performance under specified conditions of the wash liquor.
A procedure as outlined in these patent applications will consequently only be slightly better than the traditional random mutation procedures which have been known for years.
The other known techniques relate to changing specific properties, such as transesterification and hydrolysis rate (EP Publ. No. 260105 (GENENCOR)), pH-activity profile (Thomas, Russell and Fersht, supra) and substrate specificity (International Patent Publ. No. WO 88/07578 (GENENTECH)). None of these publications relates to changing the wash performance of enzymes.
A further technique that has evolved is using the detailed information on the three dimensional structure of proteins for analyzing the potential consequences of substituting certain amino acids. This approach is described in EP 260105 (GENENCOR), WO 88/07578 (GENENTECH), WO 88/08028 (GENEX), WO 88/08033 (AMGEN) and WO 88/08164 (GENEX).
Thus, as indicated above, no relationship has yet been identified between well defined properties of an enzyme such as those mentioned above and the wash performance of an enzyme.
In unpublished International Patent Application No. PCT/DK 88/00002 (NOVO INDUSTRI A/S), it is proposed to use the concept of homology comparison to determine which amino acid positions should be selected for mutation and which amino acids should be substituted in these positions in order to obtain a desired change in wash performance.
By using such a procedure the task of screening is reduced drastically, since the number of mutants generated is much smaller, but with that procedure it is only foreseen that enzymes exhibiting the combined useful properties of the parent enzyme and the enzyme used in the comparison may be obtained.
The problem seems to be that although much research has been directed at revealing the mechanism of enzyme activity, still only little is known about the factors in structure and amino acid residue combination that determine the properties of enzymes in relation to their wash performance.
Consequently, there still exists a need for further improvement and tailoring of enzymes to wash systems as well as a better understanding of the mechanism of protease action in the practical use of cleaning or detergent compositions. Such an understanding could result in rules which may be applied for selecting mutations that with a reasonable degree of certainty will result in an enzyme exhibiting improved wash performance under specified conditions in a wash liquor.
Lipases in Detergents
Examples of known lipase-containing detergent compositions are provided by EP 0 205 208 and 0 206 390 (Unilever), which relate to lipases derived from Ps. fluorescens, P gladioli and Chromobacter in detergent compositions.
EP 0 214 761 (Novo) and EP 0 258 068 (Novo) give a detailed description of lipases from certain microorganisms and disclose the use thereof in detergent additives and detergent compositions. The lipases disclosed in EP 0 214 761 are derived from organisms of the species Pseudomonas cepacia. The lipases disclosed in EP 0 258 068 are derived from organisms of the genus Thermomyces/Humicola.
A difficulty with the simultaneous incorporation of both lipases and proteases into such compositions is that the protease tends to attach the lipase.
Measures have been proposed to mitigate this disadvantage.
One such attempt is represented by EP 0 271 154 (Unilever) wherein certain selected proteases with an isoelectric point of less than 10 are shown to combine advantageously with lipases.
Another attempt is described in WO 89/04361 (Novo) which concerns detergent compositions containing lipase derived from Pseudomonas and protease derived from Fusarium or protease of a subtilisin type which has been mutated in its amino acid sequence at position 166, 169 or 222 in certain ways. It was reported that there was some reduction in the degree of attack upon the lipase by the particular proteases described.
Mutations
In describing the various mutants produced or contemplated according to the invention, the following nomenclatures were adapted for ease of reference: Original amino acid(s) position(s) substituted amino acid(s).
Accordingly, the substitution of Glutamic acid for glycine in position 195 is designated as:
Gly 195 Glu or G195E.
A deletion of glycine in the same position is designated as:
Gly 195 * or G195*
and an insertion of an additional amino acid residue such as lysine is designated as:
Gly 195 GlyLys or G195GK.
Where a deletion is indicated in Table I or present in a subtilisin not indicated in Table I, an insertion in such a position is indicated as:
* 36 Asp or *36D for insertion of an aspartic acid in position 36.
Multiple mutations are separated by pluses, i.e.:
Arg 170 Tyr+Gly 195 Glu or R170Y+G195E representing mutations in positions 170 and 195 substituting tyrosine and glutamic acid for arginine and glycine, respectively.
Further investigations into these problems have now surprisingly shown that one of the critical factors in the use of subtilisin enzymes in detergent compositions is the adsorption of the enzyme to the substrate, i.e. the material to be removed from textiles, hard surfaces or other materials to be cleaned.
Consequently, the present invention relates to mutations of the subtilisin gene resulting in changed properties of the mutant subtilisin enzyme expressed by such a mutated gene, whereby said mutant subtilisin enzyme exhibits improved behavior in detergent compositions. Mutations are generated at specific nucleic acids in the parent subtilisin gene responsible for the expression of specific amino acids in specific positions in the subtilisin enzyme.
The present invention also relates to methods of selecting the positions and amino acids to be mutated and thereby mutatis mutandis the nucleic acids to be changed in the subtilisin gene in question.
The invention relates, in part, but is not limited to, mutations of the subtilisin 309 and subtilisin Carlsberg genes and ensuing mutant subtilisin 309 and Carlsberg enzymes, which exhibit improved wash performance in different detergent compositions resulting in wash liquors of varying pH values.
Furthermore, the invention relates to the use of the mutant enzymes in cleaning compositions and cleaning compositions comprising the mutant enzymes, especially detergent compositions comprising the mutant subtilisin enzymes.
It has surprisingly been found that a decrease in the isoelectric point and hence the net charge of a subtilisin-type protease under washing conditions, can result in not only an improved wash performance of the enzyme but also an improved compatibility with lipase.
It has also been surprisingly found that compatibility of protease with lipase is influenced not only by the pIo but by the positions at which the charges are located relative to the active site of the protease: the introduction of a negative charge or removal of a positive charge closer to the active site gives stronger improvement of compatibility of protease with lipase.
Accordingly, the invention provides in one aspect an enzymatic detergent composition comprising a lipase and a mutated subtilisin protease, wherein the net molecular electrostatic charge of the mutated protease has been changed by insertion, deletion or substitution of amino acid residues in comparison to the parent protease, and wherein, in said protease, there are, relative to said parent protease, fewer positively-charged amino acid residue(s) and/or more negatively-charged amino acid residue(s), whereby said subtilisin protease has an isoelectric pH lower than that of said parent protease.